Enhancement of ultrasound-mediated gene transfection by membrane modification

✍ Scribed by Tetsuo Nozaki; Ryohei Ogawa; Loreto B. Feril Jr; Go Kagiya; Hideki Fuse; Takashi Kondo

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 231 KB
• Volume: 5
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.445

✦ Synopsis

Abstract

Background

Ultrasound‐mediated gene transfection (USMGT) with an echo contrast agent could be a new promising physical method of triggering localized gene delivery, but the effect is still modest. The aim of this study is to devise a method to improve efficiency of USMGT. We examined the effect of lidocaine and different temperatures on USMGT, each of which is a known membrane modifier, since the plasma membrane can be considered a site of action in USMGT.

Methods

We observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44 °C) on USMGT (1 MHz, 3.6 W/cm^2^ (I~SATA~) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5‐dimethyl‐1‐pyrroline N‐oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope.

Results

Lidocaine (1 mM) and heat (42–44 °C) significantly increased luciferase expression approximately 18‐fold and 19‐fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells.

Conclusions

This enhancement could be useful for ultrasound‐mediated gene therapy in the future since both treatments for membrane modification could be directly applied to the living body. Copyright © 2003 John Wiley & Sons, Ltd.